In the art of blood transfusion some special blood components, such as stem cell preparations, are frozen in liquid nitrogen for long-term storage. For that purpose, after collecting and processing the blood in standard polyvinylchloride (PVC) bloodbags, the blood components are transferred into special cryopreservation bags, designed for storage in liquid nitrogen with a temperature of -196.degree. C. When the blood components are needed for transfusion, the cryopreservation bag is taken out of the liquid nitrogen tank and thawed, usually by immersing it in a warm-water bath of about 37.degree. C. After thawing and washing the blood components, transfusion into a patient can take place.
Among the currently available cryopreservation bags the preferred one is the Hemofreeze bag, supplied by NPBI International BV in the Netherlands. It is made of a laminate film that consists of a polyamide layer laminated with a fluoropolymer layer. Such a laminate film is for instance marketed under the name of Kapton.RTM.FN by the company DuPont de Nemours. The polyamide/fluoropolymer laminate film has proven its excellence for the cryopreservation of blood for many years now in the Hemofreeze cryopreservation bags. The use of this film for blood cryopreservation bags is also described in U.S. Pat. No. 5,209,745 and WO 91/11968 of IRR et al. However, the bags in these patents are equipped with ports to transfer the blood and therefore require aseptic handling with risk of contamination. The bags cannot be connected to other bags by sterile docking, a connection technique that is regarded as sterile in the art. Sterile docking is a thermal welding process wherein tubes of different bloodbag systems are welded together while the sterility of the resulting bloodbag system is maintained. Thus, the current cryopreservation bags made of polyamide/fluoropolymer laminate film cannot be used for sterile docking as they are not equipped with the tubing necessary for sterile docking.
Other currently available cryopreservation bags are equipped with sterile docking tubing, but this is usually polyvinylchloride. Polyvinylchloride is not resistant to very low temperatures, since it becomes brittle at these temperatures. Then cracking can occur or the polyvinylchloride can break during handling in frozen condition, leading to loss of the cell preparation and contamination of the freezer. Polyvinylchloride tubing on a cryopreservation bag can therefore be used for sterile docking to transfer blood into a cryopreservation bag, but has to be sealed and removed prior to subjecting the bag to the liquid nitrogen. (Sealing tubing means that the tubing is flattened at the desired spot and both sides of the flattened tubing are sealed together, thereby closing off the tubing's fluid path). After cryopreservation, transfer ports have to be used to transfer the blood out of the bag, thus requiring aseptic handling.
However, the use of aseptic transfer equipment, such as spikes and ports, to transfer blood components between bloodbags is the most important source of microbial contamination of blood. The internal fluid path of aseptic transfer equipment is exposed to the non-sterile environment as soon as the equipment is unpacked and opened for use. After bloodbag systems are connected by means of the transfer equipment, the blood flows through this non-sterile fluid path and can become contaminated. When blood is contaminated it must be regarded as unfit for human use and should be discarded. Thus, for a safe and efficient use of blood, contamination must be prevented and the methods used in transferring blood between bags must be as safe as possible. Therefore, the transfer of blood is preferably carried out in closed bloodbag systems where the tubing and bloodbags are connected in a sterile manner. Since the internal fluid path of a closed system is never exposed to the outer environment, the risk of microbial contamination is prevented and blood can be transferred between bags without losing its sterility. Closed, sterile bloodbag systems can be provided by sterile docking, carried out with sterile connection devices such as the Terumo SCD-312. Besides maintaining sterility, sterile docking also makes blood transfer between bags easier and more efficient. Thus, in order to prevent contamination during blood transfer, sterile docking is the preferred and in fact only possibility.
Tubes of blood collection and processing bags are usually made of polyvinylchloride. For cryopreservation bags however, other tubing materials have to be used since polyvinylchloride cannot be used at very low temperatures. Thus, the tubing of a cryopreservation bag is preferably not made of polyvinylchloride. However, the tubing has to be compatible with polyvinylchloride, since sterile docking is preferred for the transfer of blood from a polyvinylchloride bloodbag into the cryopreservation bag. Compatibility implies that the tubing material has to have a melting behavior comparable to that of polyvinylchloride and that the polymer chains of the two melted materials will mix at the contact surface. This can be established by subjecting the tubing to docking experiments with subsequent tests on tensile strength and leakage of the dock. Another requirement for the tubing of a cryopreservation bag is, of course, that it has to be resistant to the temperature of liquid nitrogen. This means that the material cannot be damaged by handling it at temperatures below its glass transition temperature and that it does not lose its specific properties after being cooled down to temperatures of -196.degree. C. A third requirement for a cryopreservation bag tubing is that the tubing has the ability to be sealed by using conventional sealing technology, such as radio frequency (RF) sealing, as after transfer of the bag contents the tubing has to be closed off by sealing it.
Currently, all available cryopreservation bags lead to the same drawback: to transfer blood either to or from the bag, aseptic handling is necessary. There are no cryopreservation bags available with a sterile dockable and low temperature resistant tubing that can be used prior to as well as after cryopreservation to prevent aseptic handling and possible contamination. To overcome the drawbacks of the current cryopreservation bags, the preferred cryopreservation bag should be made out of polyamide/fluoropolymer laminate film and should have a sterile dockable and low temperature resistant tubing. The laminate film would ensure a good and proven performance of the bag at the liquid nitrogen temperature. The tubing would provide the possibility of a sterile and efficient transfer of blood between the cryopreservation bag and other bloodbags before and after cryopreservation by means of sterile docking, thus preventing contamination in the total process.
Unfortunately, such tubing cannot be connected properly to the desired film. To ensure a leaktight connection between the tubing and the polyamide/fluoropolymer laminate film, their connection should preferably be made by welding, which signifies that the heated materials are fused at their contact surface. Such a welded connection of two surfaces is called a seal. However, a sterile dockable tubing generally has a melting point that is considerably lower than the melting point of the fluoropolymer layer of the laminate film, which is the sealing layer of the film. Therefore, the required temperature for sealing the tubing to the film is too high and causes the tubing to melt before the fluoropolymer layer does. Adhesives to connect the film and tubing cannot be used either as fluoropolymers are anti-adhesive. Mechanical connections, where the materials are not fused as in a seal but are connected by a clamping force, mostly leave capillaries. These can cause leakage of fluids and gasses and therefore can cause contamination or loss of the bag contents. Therefore, a mechanical connection is only possible if it is properly leaktight. Thus although preferred, connecting a polyamide/fluoropolymer cryopreservation bag with a sterile dockable and low temperature resistant tubing without causing capillaries did not seem to be feasible up to now.